CA1320130C

CA1320130C - Methods and compositions using liposome-encapsulated non-steroidal anti-inflammatory drugs

Info

Publication number: CA1320130C
Application number: CA000539517A
Authority: CA
Inventors: Alan L. Weiner; Pieter R. Cullis
Original assignee: Liposome Co Inc
Current assignee: Elan Pharmaceuticals LLC
Priority date: 1986-06-12
Filing date: 1987-06-12
Publication date: 1993-07-13
Anticipated expiration: 2010-07-13
Also published as: ES2036593T3; EP0249561A3; EP0249561B1; EP0249561A2; DE3778972D1; GR3004723T3

Abstract

METHODS AND COMPOSITIONS
USING LIPOSOME-ENCAPSULTED NON-STEROIDAL
ANTI-INFLAMMATORY DRUGS

ABSTRACT

Methods and compositions are described for the treatment of inflammatory diseases including the use of liposomes to deliver nonsteroidal anti-inflammatory drugs. Drugs may be encapsulated in the liposomes during their preparation, or alternatively, are combined with the liposomes following their formation. The composition may include glycolipids such as galactolipids including digalactosyl diglyceride, and the liposomes may be made by a number of procedures and rendered free of exogenous nonsteroidal anti-inflammatory drugs. The compositions may be administered to mammals including humans.

Description

13~0130 THODS~AND COMPOSITIONS
USING LIPOSOME-ENCAPSULATED NON-STEROIDAL
ANTT-I~ AMMATORY DRU~S

FIELD OF T~E INVENTIO~

The pre~ent invention ls directed to the ereatment of diseaqe tates, such a~ inflammation, paln and fever, using composition~
comprising a non-~teroidal anti-inflammatory drug which may be encapsulated ~n a liposome. More particularly, the in~ention describes methods for reducin~ toxic side effects related to non-steroidal anti-inflam~atory drugs by ~dministering these drugs in liposome~, wherein the compoqition alqo include~ a glycolipid.

In a preferred embodiment this invention relates to the novel concept of substantially eacapsulating nonsteroidal anti-inflammatorY
drugs or therapeutic agents and analogues and derivative~ thereof in ga~tric resistant llposome~ to reduce ~astrointestinal irritation associated with the adminlstratio~ of such therapeutic agent~ to mammal~
including human~.

The preferred compo~ition and methods described herein proYide gastric reqistant liposomes that contain encapsula~ed nonsteroldal 20 : anti-inflammatory therapeu~ic agent~ and analogues and derivative~
thereo~O These compositions are primarily free of exo~enous nons~e~oidal anti-in~lam~a~ory therapeutic ~gents previously asqoc~ated wit~ ~herapy regimen3 and partlcularly with~chronic therapy re~imens (in excess of bsut three or four days), thuY minimizin~ ~astrointeqtinal insult.

~2-:~ : :
: . : :
: : : , , . - :

~: :

~ 320130 BACKGROUND OF THE I~VENTION

gecent pharmacological research has disclosed a group of anti-inflammatory therapeutic agents which are not steroidal ln character. These agents may generally be classified as carboxylic acids Sincluding salicylates, acetic acids, propionic acids and fenamates), pyrazoles, and oxicams).

While these therapeuti agents have been widely accepted they share a general characteristic of potentially being associated with gastrointestinal irritation and lesions. For example, indomethacin, a widely used agent, which i3 an acetic acid sal~, is known to be assoclated with gasrointestinal ulcers. See, e.g., the work o~ Miklos Ghyzy et al. in U.S. Patent ~OB. 4,378,354 and 4,421,747. These patents disclose improvement in reducin~ non~teroidal anti-inflammatory associated ulceration by admixing nonsteroidal anti-inflam~atorie~ with phospholipids. Liposomes are generally known but condition~ necessary to avoid or minimize nonsteroidal anti-inflammatory associated gastrointestinal insult have remained unidentified. See, e.g., Science 219:1327-1329 March 19, 1983 "Role of Surface-Active Phospholipids in Gastric CytDprotection," Lichtenberger et al.

Liposomes are completely closed bilayer membrsnes containing an entrnpped aqueous volume. Liposomes may be unilamellar vesicles (posse~sin~ A single membraTIe bilayer) or multilamellar vesicles (onion-like structures characterlzed by multiple membrane bilayers, each separated from the next by an aqueous layer). The structure of the resulting membrane bilayer is such that the hydrophobic (non-polar) "tail~" of the lipid orient toward the center of ~he bilayer while the hydrophilic (polar) "heads" orient towards the aqueous phase.

The original liposome preparation of Ban8ham e~. al. (J~ Mol~
Biol., 13, 238-252 1965) involv s su~pending phospholipids in an organic solvent which i9 then evapora~ed to dryness lea~ing a phospholipid film on the reaction vessel. Then an appropriate amount of aqueous phase is added, the mixture is al]owed to "swell", and the resulting li~ ~s ~ ~sl 3 0 which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. This technique provides the basis for the development of the small sonicated unilamellar vesicles described by Papahadjopou]os et al. (Biochim. Biophys. Acta. 135, 624-638 1967), and large unilamellar vesicles.

Other techniques that are used to prepare vesicles include those that form reverse-phase evaporation vesicles (REV), Papahadjopoulos et al., U.S. Patent No 4,235,871, stable plurilamellar vesicles (SPLV), Lenk et al., U.S. Patent No. 4,522,803, monophasic vesicles (MPV), Fountain et a]., U.S. Patent No. 4,588,578 and freeze and thaw multilamellar vesic]es (FATMLV), Bal]y et al., PCT Application No.
86/01371, published January 15, 1987.

In a liposome-drug delivery system, the medicament is entrapped in the liposome and then administered to the patient to be treated. For example, see Rahman et al., U.S. Patent No. 3,993,754; Sears, U.S. Patent No. 4,l45,410; Papahadjopoulos et al., U.S. Patent No. 4,235,871;
Schneider, U.S. Patent No. 4,224,179; Ienk, et al., U.S. Patent No.
4,522,803; and Fountain, et al., U.S. Patent No. 4,588,578.

References cLting the use oE phospholipids to treat gastrointestinal ulceration have appeared in the literature; this effect due to the purported abiLity of the lipid to restore the stomach's natural protective barrier to irritants. For examp]e, Dia] et al., Gastroentero]ogy, 87, 379-385 (1984), suggested the anti-ulcer activity . .
oE bovine milk was due to its concentration of dipalmitoyl phosphatidy]choline. Other studies cite lysolecithin (Clemencon et al., Scad. J. Gastroenterol. Suppl., 19, 116-120, 1984) and lipids isolated ...... _ ....... _ . _ from both the fruits of the Melia azedarach plant, and the mussel Perna canaliculus (Al-Khatib, Jpn. J. Pharmacol., 36, 527,533, 1984, and ......
Rainsford et al., Arzneim.-Forsch., 30, 2128-2132, 1980, respectively), as ulceroprotective agents In rats.

.. . .

.

.

~01~0 Another study assessing membrane damage incurred by ~odium dodecyl sulfate cites phosphatidylcholine (Martin et al., J. Pharm. Pharmacol., 33~ 754-759, 1981) as a protective agent agalnst such damage. Flnally, Lichtenberger et al. (Sciencer 219, 1327-1328, 1983) studied the ameliorative effects o~ a liposomal pho~pholipld suspension composed of 135 ug of dipalmitoyl pho~phatidylcholine, and 15 ug each of phospha-tidylethanolamine, phosphatidylglycerol, phosphatidylinositol, and sphingomyelin. They postulate the enhanced protection due to formation of an absorbed hydrophobic layer between the gastric epi~helium and the lum~nal content~. Prostaglandins have been clted a~ protectants against gas~ric ulcerogEnesis and ble~ding in laborato~y animals and man (Robert et al., ~astroenterolo~, 77, 433, 1979, and Robert et al., Gastroenterolo~y, ~5, 481, 1968), however Llchtenberger et al. (vide lnfra) determined that prostaglandin synthe~is was not required for extrinsic phospholipid-induced ga~tric protection. Ho~ever, when rats were dosed with Pro~taglandin E2, rat stomach mucosa demonstrated a 2-6 fold increa~e in the ma~or gastric sur~ace protective surfactant with the greate~t enhancements seen in concentrations of phosphatidylethanolamine and phospha~idylcholine.

In addition, I,ichtenberger et al. European Pat. Appl. 92121, published October 26, 1983, suggest phospholipid compositions for the treatment of ulcer. An application for similar compositions combined with a prostaglandin, for treatment of ga~tric and intestinal ulcers is Imsgawa et al., European Pat. Appl. 150732, published August 7, 1985. A
further reerence suggesting anti-ulcer composition~ i~ Amen et al., U.S.
Patent No. 4,029,773, for a saccharose, amino acid and trigylceride mixture.

Ghyczy et al., U.S. Patent ~o. 4,$28,193, dlsclose~ compo~itions and methodq of treating inflammation comprising phospholipids and non-steroidal anti-inflammatory drugs where the molar ratio is about l:O.l to 1:20. The mi~ure i~ prepared by co-solubiliz~ng the drug and lipid iD organic solvent, followed by removal of the solvent by .

.. , ., . ., . ~ . .

~ 1320~30 distillation. Alternatively, the components are co-mixed in water. The Qolution~ ~o obtained are then lyophilized.

A ma~or plant galactolipid, digalactosyl diglyceride (DGDG) has been u~ed to prepare liposomes. DGDG accounts for about 40% of the total S lipid content of higher plant chloropla~t and thylakoid membranes (Quinn et al., Pro~. Biophvs Molec. Biol. J 34, 109-173, 1978). It has been used in studies where photo~ystems utilizing chlorophylls and cytochromes are reconstituted into liposomes (Sprague et al., J. Cell Biol., 100, 552-557, 1985, and Mor~chel et al., J Cell Biol. 97, ~01-310, 1983, respectively). Studies invol~ing immunological activity of DGDG in liposomes as measured by complement dçpendent gluco~e release (Alving et al., ImmunocheMistrv, 11, 475-481~ 1974) and the reactiYity of 3era from multiple sclerosis patients with DGDG llposome~ and its ability to cause complement-mediated lysi~ of the liposome~ ~Boggs et al., J. Neurol.
Sci., 66, 339-348, 1984) have been performed. DGDG ha~ been suggested as a minor liposome component for the purpose of delivering liposomal-encapsulated drugs to hepatocytes (Geho, U.5. Patent No.
4,3779567~.

There is an ongoing need for compositions which can buffer the unwanted gastrointestinal ~ide effects of MSAIDs.

It i~ an object of thl~ invention to provide a form of non~teroidal anti-inflammatory therapeutic agent including analogues and derivative~ thereof that i~ administerable with minimized gastrointe~tinal ulceration or irritation.

It is a particular obJect of this inventlon to provide a fonm of nonsteroidal anti-inflammatory therapeutic agent including analogues and derivatives thereof that is admini~terable r~ 09 and does not cause 8a trointe8tlnal ulceration or irritation.

It is a further ob~ect of this inventlon to provide a gastric resistant lipo~ome preparation containing non~eroidal anti-inflammatory .
`
- ` ~

-" ~3~0~ 30 agents and analogues and derivatives thereof wi~hin the lipo30me, yet has limited or no exogenou~ nonsteroidal anti-inflammatory agent pre~ent.

It i8 another ob~ec~ of this invention to provide a gastric resistant liposome tha~ when admlnistered to an animal releaQes nonqteroidal anti-inflammatory therapeutic agent in a controlled manner.

SUMMARY OF T~E INVENTION

The pre~ent inven~ion presents new and surpri~ingly nonirritating preparation~ for admini3tering nonsteroidal anti-inflammatory therapeutic agents to mammal.~ including human~. The ~erm nonsteroidal anti-inflammatory therapeutic agPnt~ ~hall be understood to include the analogue~ and derivatives of ~uch agents.

Furehermore, particular compo3ition~ are provided for non-steroidal anti-inflammatory drugs with a glycolipid. The glycolipid can be a glyco3phingolipid or a galactolipid, such a~ digalacto3yl diglyceride. The pharmaceutical composition may be a lipo-qome composition compo~ed of the above-mentioned glycolipids.

In addition, this invention compri_es the preparation and uqe of ~a~tric resi~tant liposome~ contalning non~teroidal anti-inflamatory therapeutic agents, wherein the preparations are primarily free of exogenou~ nonsteroital anti-inflamatory therapeutic aBents.

Pr~ferred lipo~omes of this invention are prepared from both un~aturated lipids, saturated lipids and mixture~ thereof. Saturated lipldo do not contain carbon-carbon double bonds on the long chain fatty acid component of the lipid. Un~aturated lipidq can be hydrogenated to obtain ~aturated lipids with carbon-carbon double bonds removed.
Alt~rnatively, saturated lipids can be 3ynthesized usin6, for example, aturated long chain atty acid~. ~
: ~

: `: :: :

: ~ : : :

:

`

.

-~ ~32~130 Unle~ otherwi~e differentiated, the terms saturated and hydrogenated are u~ed ~ynonomously herein to mean a lipid which doe3 not contain a carbon-carbon double bond in its long chain fatty acid portion.

~hi8 invention lncludes a composition compri~ing a therapeutically effective amount of at lea~t one non~teroidal anti-inflammatory therapeutic agent primarily encapsulated in a gastric resistant lipo~ome and primarily ab~ent exogenous non~teroidal unti-inflammatory therapeutic agent. Preferred aspect~ of the composition are (1) lipo~ome~ comprised of eg8 phosphatidylcholine or digalac~osyldiglycer~de, (2) lipo~ome~
sub3tantially comprised of a lipid which 1~ sat~rated 3uch as hydrogenated phosphatide3 such a9 hydrogenated egg or ~oy phosphatidylcholine (3) indometharin as a non~tProidal anti-inflammatory therapeu~ic agent, (4) as to the total weight of non~teroidal anti-inflammatory therapeutic agent, ~he exogenou~ percentage thereof is less than 30% with le~ than 25% being more preferred and le~ than 5%
further preferred, (5) liposomes of 3ubstantially equal solute distribution such as monophasic vesicles or frozen and thawed multil~nellar ve~icles being more preferred while stable plurilamellar veRicles are preferred for lipo~omes substantially of saturated lipid, and (6) the composltion adapted for oral administration. Further included are compositions wherein the nonsteroidal anti-inflammatory therapeutic agent is selected from salicylates, acetic acids, priopionic acids, fenama~es, pyrazoles, oxicams and analogues and derivatives thereof.

~li9 invention also includes a pharmaceutical dosage form comprising at least one nonsteroidal anti-inflammatory therapeutic agent primarily encapsulated in a gastric resi3tant llposome and primarily ahsent exogenous nonsteroidal anti-inflammatory therapeutic agent in an acceptable pharmaceutical carrier or diluent. Preferred aspects of the pharmaceu~ical dosage form include tha~, as to the total weight of non~teroidal anti-inflam~atory therapeutlc agent, the exogenous - percentage ~hereof is le~3 than 30% with le~s than 25% being more preferred and less than 5~ further preferred. In the mo~ preferred ,., ~, ~ .

`~, 132~130 embodlment the pharmaceutical dosage form i~ adapted for oral ad~inistration, particularly, with unsaturated lipid liposome3, a3 a monophasic vesicle or a frozen and thawed multilamellar vesicle and furthçr where either vesicle type contain~ indomethacin. Similarly, the preferred dosage form for liposomes substantially of saturated lipid is the stable plurilamellar vesicle form. Further included are compo~itions whereln ~he nonsteroidal anti-inflammatory therapeutic agent is 3elected from salicylate~J acetic Arids, pr~opionic acids, fenamate~, pyrazole~, o~icams and analogues and derivatives thereof.

This invention also includes a method of minimizing the ga~trointestinal irritation a~ociated with the administration of a therapeutically effectiYe dose of at lea~t one non~eroidal an~i-inflammatory therapeutic agent to a mammal including a human compri~ing admini~terin8 said non~teroidal an~i-inflammatory therapeutic a8ent to said mammal in the form of a gastric resistant llposome which prlmarily encapsulates ~aid nonsteroidal anti-inflammatory therapeutic agent and in which exogenous non~teroidal snti-inflam~atory therapeutic agent is primarily absent. Preferred aspects of the method are (1) using preparations wherein as ts the total weight of nonsteroidal an~i-inflammatory therapeutic a8ent the exogenous percentage thereof is less than 30~ with le~ than 2S% being more preferred and less than 5%
further preferred, (2) using liposomes of 3ubstantially equal solute distribution particularly monophasic Yesicle~ and frozen and thawed multilamellar vesicles, and as to lipoRome~ substantially of saturated lipid using stable plurilamellar veslcles and (3) uqin~ indomethacln as a nonsteroidal anti-inflammatory therapeutic agent and (4) employing the method in the oral admlnistration of the composition. Further included are compo~itions wherein the nonsteroidal anti-infla~matory therapeutic agent i9 selected from salicylates, acetic acids, prlopionic acid~
fenamate~, pyrazoles, oxicam~ and analogues and derivatives thereof.

BRIEF DESCRIPTIO _~F THE DRAWING

FIG 1. Fractionation of indomethacin-containin8 EPC MP~ by sucrose gradient centrifugation. Nonophasic ve~lcles cons~ructed with - _g_ .

`` 132~13~
100 mg of egg phosphatidylcholine, 0.25 uCl of 1 C-indomethacin, and either 15, 17, 19, 21, 23, or 25 m8 of indomethacin were loaded snto linear 5-20% ~ucro~e gradients and centrifuged at 288,000 xg for 2.5 hour~.

FIG. 2 Comparative plasma level~ of therapeutic agent obtained using saturated and unsaturated lipid liposomes.

DETAILED DESCRIPTION OF T Æ INVENTION

The following abbreviations will be employed:

SPLV - stable plurilamellar ve~icle : MLV - multilamellar vesicle MGDG - mono~alacto~yl diglyceride DGD~ - d~galacto3yl diglyceride CHS - cholesterol hemi~uccinate ~SPC - hydrogenated soy pho~phatidylcholine THS - tocopherol hemisuccinate MPV - monopha~ic vesicle FATM4V - MLVs produced by a freeze and thaw technique T - ve9icles formed by one or more extrusion~ through filter apparatus REV - reverse phase evaporation vesicle NSAID - non~teroidal anti-inflammatory drug The nonsteroidal anti-inflammatory drugs are a highly effective group of dxugs) however, their use is limited due to their toxici~y. We have found that administering ~S~IDs encapsulated in liposomes reduces gastrointestinal side e~fects including ulceration while maintaining their efficacy.
The non~teroldal anti-inflammato~y therapeutic agents employed in thls invention include by way of example:
: ~

::

.

..

:; ~ ' '" : .
` ' '. ~ ' ' ' ' .

l 320130 Carboxylic acid~
Salicylate~
Acetylsalicylic Acid (ASA) Sal~alate Difluni~al Fendosal Acetic Acids Indomethacin Acemetacin Cinmetacin Sulindac Tolmetin Zomepirac Diclofenac Fenclofenac I~oxepac Furofenac Fen~ia~ac Clidanac Oxepinac Fenclorac Lonazolac Metiazinic Acid Clopirac Amfenac Benzolfenac Clometacine Etodolac Bumidazone Clamidoxic Acid Propinonic Acids Ibuprofen Flurbiprofen ~aproxen Ketoprofen Fenoprofen Benoxaprofen Indoprofen Pirprofen Caprofen Oxaprozin Pranoprofen Suproen Microprofen Tioxaprofen :: ~ Alminoprofen Cicloprofen Tiaprofen~c Acid Furaprofen : Butibufe~
~ ; Fenbu~en : ~ : : ::: : :

: ~:

:
' .

~urobufe~ ~32013Q
Buclo~ic Acld Protizi~ic Acid Fen~mate~
Mefanamic Acid FlufenAm~c Acid Meclofenamate ~iflumic Acld Tolfenamic Acid Fluni~in Clonixin Pyrazoles Phenylbutazone and AnalogQ
Peprazone (Prena~one) Apazone (Azapropazone) Tri~e~hazone Mofebutazone ~ebuzone Su~ibuzone Oxicam~
Piroxicam Isoxicam Tenoxlcam Useful in lipo~ome preparatlon are lipids that reQul~ in a bilayer such that a hytrophobic portion of the lipid orients toward the bilayer while a hydrophilic portion orients toward an aqueouQ phaAe.

Lipids that may be used in thc ~resen~ lnvention include glycolipids such a~ glycosphingolipids a~d ~alactolipids ~uch a~ dlgalacto~yl diglyceride (DGDG) or mono~alacto~yl diglyceride (MGDG) and DGDG and/or MGDG in combination with phospholipids such as phosphatidylcholine, phosphati~ylserine, pho3phdtidylino3itol, or pho~phatidylethanolamine and their derivative~ and sterol or tocopherol monoesters of diacids, such as cholesterol he~i~uccinAte and tocopherol hemisuccinAte, re~pectively.

Glycolipid~ that m~y be used i~ formin~ the vesicles of ~he inventlon include glycosphingoliplds and galactolipld~ ~uch as monogalactosyl dlglyceride (~GDG) or digalactosyl diglyceride (DGDG), preferably DGDG. DGDG occurs in n&ture a~ a plant lipid in chloroplasts 15 : and ha~ the structure: r~
s ~

~ -12-: :: :

' ,~

. ' .
, 3 ~

wherein each R substituent i9 in the C15-C17 chain range, ca 20~
palmitic, 9% oleic, 66% linoleic, the balance being stearic, linolenic, and other fatty acids. (Myhdre, Can. J. Chem., 46, 3071-77, 1968~

The MGDG molecule has a ~ingle neutral galactose residue on its polar headgroup. Biophysical studies (13C-longitudinal relaxation ti~e~) imply that the MGDG headgroup is ~ignificantly ~maller than ~hat of other lipids ~uch as DGDG and phosphatidylglyoerol. Hence, the molecule has a cone shaped structure, with ~he interactions of the highly unsaturated acyl chain~ giYiD8 ie a relatively bulky hydrophobic region.
0 Thu9 MGDG doe~ not form lamellar structures but forms a hexagonal-II
structure under hydratio~. Mixtures of MGDG with bilayer forming lipid~
will adopt a bilayer structure at concentrations of MGDG up to about 60 mol %. Eligher proportions of MGDG will result in lipidic par~icles and other nonbilayer ~tructures.

DGDG has an additional ealactose unit on the polar headgroup compared ~o MGDG, thereby giving i~ a relatively bulky headgroup, but it also has bulky hydrophobic acyl chains. Structurally, DGDG fo~ms cylindrical-3haped structures and induces bilayer organization in membranes. Thi~ feature makes DGDG the preferred galactolipid of the two for the formation of liposomes. In combined aqueous dispersions, MGDG
and DGDG form mixed l~mellar and inverted micelle phases at a 2:1 weight ratio. Studies ha~e been undertaken to form lipo30mes with MGDG and DGDG
combinations using detergent solubilization techniques with Triton X-100. U~ing this technique, bilayer ~tructures may be ~ormed using both ~alactolipids only up to a MGDG:DGDG weight ratio of 20:~0. Hex tubes formed in preparations using 30% or greater MGDG. Mixtures containing equal ~ei8hts of MGDG and DGDG produce ~tructures with only traces of bilayer areas containing lipidic particles.

Phospholipid3 are al80 useful. Lipids may be utilized alone or in 3Q combinaeion. Preferred lipids that offer "gastric resistance" ~ore fully descrlbed below) in&l~de thc phosphatldes, phosp~atidylcholine, ':

-` 132~
phosphatidylethanolamine, phospha~idylglycerol, as well as sphingomyelln. Particularly preferr~d in dosage applications requiring a controlled elaboration of nonsteroidal anti-inflammatory therapeutic agent are liposome3 substantially comprised of saturated or hydrogenated lipids, par~icularly phosphatides and most particularly hydrogenated egg or soy phosphatidylcholine. The controlled elaboration of therapeutic a8ent provided by liposomes comprised qubstantially of saturated lipid is distinct in avoiding a peak level of therapeutic a8ent essentially coincident with administratlon. This fact can be observed from the blood levels of indomethacin associated with administration of hydrogenated soy phosphatidylcholine orally administered to rats, FIG.2. Peak blood levels have been associated with the contraindications of nonsteroidal anti-inflamatory ~herapy by 30me inYestigator~.

The term "substantially" referring to th~ amount of saturated lipid comprlsing such a lipo~ome refers to at least about 1:1 saturated lipid to unsaturated lipid bssed upon the molar ratios of the lipids with about 9:1 saturated to unsaturated belng more prefered and essentially all lipid fully saturated being mos~ prefered. Liposomes of satursted lipids may be admixed with other lipids, particularly cholesterol.

Certain lipids are amphipathic only when in the form of a salt and not in the acid state and thus do not form gastric reslstant liposomes.
Cholesterol and tocopherol hemisuccinate3 ("CHi" and "THS," respectively) are exemplary of such materials. Upon exposure to conditions co~mon in the gastrointestlnal trac~, the amphipathic salt form of 3uch materials returns to the aqueous soluble acid form and liposomes b~sed upon these materials rapidly break down. Thi~ breakdown occurs within seconds of contact with the low pH associated with the ga~tric en~ironment. In the practice of a preferred aspect of thi3 invent~on only gastrir resistant liposomes may be utilized. Thus, gastric resistant 1iPOQOme8 may be defined as those lipo~omes prepared from lipid~ excluding tho3e lipids that cease to be stable in a bilayer structure upon exposure to pH
conditions common in the ga3trointe~tinal tract. Ga tric re~istant lipo~ome~ of improved stability may be made from hydrogenation or 3aturation of unsaturated lipids.

-.

132~3~

The liposomes that may be used in the invention include, but are not limited ~o MLVs, small or large unilamellar vesicles (SW8 or L Ws, respectively), V~Ts and those having equal solute distribution, such as SPLVs, MPV3, and FATMLVs.

A variety of methods may be used to prepare a liposome composition comprising an NSAID and a galactolipid, such as DGDG. In one method, the NSAID is combined with the lipid in organic solvent, the solution rotary evaporated to a thln film and finally, the film hydrated wlth an aqueous medium such as aqueous buffer, forming liposomes. Such a procedure forms 1 0 MLV~ .

Another method for preparing ~SAID-galactolipld liposomes is to comhine the ~SAID with the galactolipid in organic solvent, rotary evapora~e the solution to a thin film, and dissolve the dried film in ethanol to which has been added an aliquot of an aqueous medium 3uch as aqueous buffer. Thi~ solution is ~hen rotary evaporated to a thin film, and the film then hydrated wi~h aqueous medium, forming liposomes. Such a procedure forms MPV~.

Yet another method for preparing NSAID-galactolipid liposomes i9 to combine the NSAID with the galactolipid in organic solvent, rotary evaporate the solution to a thin film, a~d resuspend the ilm in diethyl ether. A small aliquot of an aqueous medium such as aqueous buffer is then added to the organic solvent ~olution, and this solution is dried under nitrogen ~as to a paste while sonicating in a bath sonicator. The paste is then hydrated with an agueous medium, forming liposomes. Such a procedure forms SPLVs.

A further methcd for preparing NSAID-galactolipid liposomes is to form MLVs a~ described aboYe, then sub~Pc~ these MLVs to a number of freeze and thaw cycles. Such cycle3 are carried out by first rapidly cooling the MLV suspension to obtain a frozen lipid-aqueous medium mi~ture, and then warming the mlxture. The freezing and warming step~

.. . .

~320130 are pre~erably performed at ]east about five times. Such vesicles have an equal solute distribution and are known as FATMLVs.

Yet another method Eor preparing NSAID-galactolipid liposomes is to form MLVs as described above and extrude these liposomes through a filter under pressure of about 700 psi. Such resulting vesicles are known as VETs and can be prepared according to the procedures of Cullis et al., PCT publication WO/86/00238 on January 16, t986.

Within the class of useful liposomes is a preferred subclass of ~liposomes characterized in having solu~e distribution substantially equal to the solute distribution environmen~ in which prepared. This subclass may be defined as stable plurilamellar vesicles (SPLV) as defined in U.S.
Patent No. 4,522,803 to Lenk et al. and further includes both monophasic vesicles described in U.S. Patent No. 4,588,578 to Fountain et al. and frozen and thawed multilamellar vesicles (FATMLVs) as described in "Solute Distributions and Trapping Efficiencies Observed in Freeze-Thawed Multilamellar Vesicles" Mayer et al. Bioc ~ Acta ôl7:193-196 (1985). Liposomes specifically prepared by the process of U.S. Patent Ser. No. 4,522,803 are referred to as stable vesicles. It is believed that the particular stability of the SPLV type liposomes arises from the low energy state attendant to solute e.quilibrium.

Both CHS- and THS--containing vesicles may generally be prepared by any method known to the art for preparing vesicles. In particular, see the procedures of the copending patent applications of Janoff et al., PCT
Patent Application No. 85/00631, entitled "Steroidal Liposomes", pub-lished bctober 24, 1985, Janoff et al., PCT Patent Application No.
85/01983, entitled "Steroidal Liposomes", published October 23, 1986, and Janoff et al., PCT Patent Application No. 86/02101, entitled "Alpha-Tocopherol Based Vesicles", published April 23, 1987, respectively. Ac-cordi~ng to these procedures, the powdered forms of sterol or tocopherol monoesters of diacids, such as CHS or THS are added to an aliquot of aqueous buffer, and vortexed to fully suspend the dispersions forming MLVs. The dispersions are then sonicated in a water bath for several hours forming SUVs, and the drug powders added directly to these soni-, .

-, ~32~
cated vesicles, and vortexed to fu]ly disperse.

Another technique for forming MGDG/DGDG liposomes is to solubilize both lipids below 20 C in a fluorinated hydrocarbon (such as *Freon-22) below the boiling point of the fluorinated hydrocarbon. An aqueous medium is then added forming an emu]sion. The emulsion is then warmed above the boiling point of the fluorinated hydrocarbon (e.g. 20C), to remove the fluorinated hydroca-rbon, resulting in liposome formation.

In combined dispersions of MGDG and DGDG used to form reverse phase evaporation vesicles (REVs), bilayers accommodate slight]y greater amounts of MGDG before major surface irregularities appear, as compared to the detergent dialysis technique. Liposomes formed using a 40:~0 weight ratio of MGDG:DGDG contain hex-II tubular arrays in bilayer vesicles. Clumping of the lipids in the aqueous phase does not occur until 70% MGDG is reached. Both MGDG and DGDG may be successfully combined with other lipids such as phosphoLipids, sterols such as cholesterol esters, or phenols such as tocopherols to form liposomes.

A liposome preparation can also be composed of a combination of CHS and TllS, or other organic acid derivatives of a sterol and a tocophero].

Where necessary, as in the SPLV and MPV procedures, organic sol-vents may be used to so]ubi]ize the Lipid during vesicle preparation.
Suitable organic so]vents are those with a variety of polarities and dieLectric properties, including ch]oroform, acetone, methylene chloride, diethyl and petroleum ethers, and mixtures oE chloroform and methanol.

Liposomes entrap an aqueous medium which is enclosed by the lipid bi]ayers. The aqueous medium can be for example, water or water containing a dissolved salt or buEfer. Examples of such salts or buffers can be sodium chloride and phosphate buffered saline (PBS). Other buffers include but are not limited to Tris-HCl (tris-(hydroxymethyl)-aminomethane hydrochloride), and HEPES

* Trade-mark ' ' -, 132~
(~-2-hydroxyethyl p~perazine-N'-2-ethane sulfonic acid). Buffers may be in the pH range vf between about 5.0 and about 9.5. In the preferred embodiment, tbe preparations are hydrated with phosphate buffered saline (PBS) at pH of between about 5.0 and 9.5, preferably about pH 7.4. In the case of CHS- and THS-containing ve~icles which employed the Tris salt form~ of CHS and T~IS, a Tris/HCl buffer at pH of about 7.4 was used.

The SPLV Process compri~es forMing a solution of at lea~t one gastric resistant lipid in at lea~t one organic ~olvent plus a first aqueous component containing at least one nonsteroidal an~i-infla~matory therapeutic agent. Sufficient aqueous component to form a monopha~e is utilized. The next step is evaporating organic solvent from the monophase at tempera~ure and pressure which maintalns and farilitates evaporation until a film forms. The next step is adding a ~econd aqueous component to the film and agitating the second aqueous component with the film in order to resuspend the film and to fo~m lipid ~esicles. The resulting material may be washed of exogenous nonsteroidal anti-inflammatory therapeutic agent as required. This method i3 further described in Example 4 employing indomethacin but it is to be under~tood to apply similarly to other nonsteroidal anti-inflammatory therapeutic aBents as well.

The preferred gastric resistant liposomes of this in~ention containing a non~teroidal anti-inflammatory therapeutic agent or combinations of ~uch agents are deemed to primarily free of exogenous nonsteroidal anti-infl G atory therapeutic aBents when ~uch exogenous mater~al comprises less than about 30~ of the total ~eight of nonstexoidal anti-infla~atory therapeutic agent or agents present. In the preferred preparation less than about 25% of to~al nonsteroidal anti-in~lammatory therapeutic agen~ is exogenous. The threshold dosage of nonsteroidal anti-infla~matory therapeutic agents causing ga~trointestinal irritation and gross gastrointestinal da~age (such as uleeration) will Yary with a number of fac~ors includlng the nonsteroidal anti-inflammatory therapeutic agen~, the method Gf administration, the dosage and duration, the mammal and it~ presentlng conditlon. In .

. . . ' ' ' .

~al30 general, the most preferred preparations haYe the least exogenous nonsteroldal anti-infla~matory therapeutic agent and these preparatlons characteristically have exogenous nonsteroidal anti-inflammatory therapeutic agent of less than about 5% of the total weight of nonsteroidal anti-inflammatory therapeutic agent or agents present and some less than 1%. A further consideration in use of this invention will be the half-life of the nonsteroidal anti-inflammatory therapeutic agent in the blood. While not being bound to any particular theory, it is thought that the nonsteroidal anti-inflnmmatory therapeutic agents with longer half-live~ have a greater propensity to cause gastrointestinal in~ult. Of course, this factor may be counter-balanced by other factors ~uch as those noted above. Indomethacin, tolmetin, ibuprofen, diclofenac, fenoprofen, acetylsalicylic9 and meclofenamate have generally been shown by others to have blood level half-lives under four hours.
Mefenamic acid and flurbiprofen have generally been shown by others to have blood level half-lives of about four and 5iX hours respectively.
Naproxen, difluni~al, sulindac, piroxicam and phenylbutazone have generally been sho~n by others to have half-lives from over ten hours ~o three days. The degree of amelioration of ga~trointenstinal irritation by the product and method of this invention is anticipated to vary as to each nonQteroidal anti-inflammatory therapeutic agent bu~ will likely be more pronounced as to nonsteroidal anti-inflammatory therapeutic agents with relatively shorter half-lives.

It has now been determined that liposomal encapsulated non~teroidal anti-inflammatory therapeutic agent, primarily free of exogenous nonsteroidal anti-inflammatory therapeutic agent, caused considerably less gastrointestinal in~ult than the administration of equivalent doses of lipid and nonsteroidal anti-inflammatory therapeutic agent as mere admixtures coadministered. The latter condltlon clearly comprise~ the limitinB situation wherein virtually all of ~he nonsteroidal anti-inflammatory therapeutic sgent is exogenous.

In preparing gastric resistant MPV liposome~ of this in~ention, an aliquo~ each of lipid and nonsteroidal anti-inflsm~atory therapeutic ~ 1320130 agent are codissolved into absolute ethanol at ratios pr~ferably exceeding about 6:1 by weight (lipld:nonsteroidal anti-inflammatory therapeutic agent). Depending upon various conditions including both the solubillty of the nonsteroidal anti-inflammatory therapeutic agent and lipid ratio3 from generally about 1 ml ethanol per 100 mg lipid may be used. For convenience in the laboratory 5 ml of ethanol i9 preferred per 200 m8 lipid.

An aqueou~ componene is added to the ethanol organic solven~ in a ratio with the ethanol of from about 25:1 to about 1:1 (solvent:aqueou~). The re~ultlng monopha~e i9 placed under rotoevaporation and at reduced pre~sure until a film forms. The film may be rehydrated to any de~ired concen~ration although 200 mg lipid per ml aqueous solution i~ preferred. Thi~ method may be utilized with salicylates, acetic acids, propionic aclds, fenamates, pyrazoles, oxicams and analogues and derivatives ~hareof.

As each non~teroidal anti-inflammatory therapeutlc agent will present a particular set of physical characteristics including a range of solubilities, lipo~omal entrapment ranges, and deQired dosages, a preferred test procedure for determining entrapped/exogenous nonsteroiodal anti-inflammatory therapeutic agent i9 presented in Example 6, but other suitable te~t procedures are also well known in the art and~are useful.

To determine the relative amounts of liposomally entrapped exogenous non~teroldal anti-inflammatory therapeutic agent a number of procedures well known to those skilled in the art may be used. A method of radiolabeling A sucrose ~radient centrifugation was employed.
.
In the radiolabel procedure radiolabeled non~teroidal an~i-inflammatory i9 supplPmented into the lipid solvent and a small amou~t of unl~beled aqueous material is thereafter al~o added. Th~
solvene 1~ then removed by any con~enient method such as rotoevaporation. For ethanol, roeoevapora~lon is convenlently performed ~ 20-:

' 320~30 at elevated temperature of about 50C and reduced pres3ure such as from about 1 to lO0 mmHg.

Those skilled in the art will clearly understand the appropriate reaction conditions for other solvent~ and nonsteroidal anti-inflammatory therapeutic a8ents.

After solvent removal, the material i~ resuspended in suitable aqueous ~olution such as saline.

The resulting liposomes may then be e~amined for drug loading. This is done by separating liposomes from nonlipo~omal material by any separating means. Sucrose gradien~ eparation i~ one such means.

If a sucrose column i9 used, the column iQ centrifuged and thereafter fraction~ collect and counted in a ~cintillation counter.
Radiolabel at the bottom of ~uch column represent~ exogenou~ nonsteroidal anti--inflammatory and the fraction a~ the top represents liposomally encapsulated nonsteroidal anti-inflammatory therapeutic agents. The percentages exogenous and encapsulated may then be calculated.

Results of te~ts of the eg8 phosphotidylcholine (EPC)tindomethacin liposomal preparations made by the method of Example 4 which contain increasing level~ of lndomethacin demon~trate that at weight ratios below approximately 6:1 pho~pholipid:drug re~pectively, hlgh density exogenou3 material containing nonsteroidal anti-inflammatory crystals becomes apparent (FIG. 1). Ae lower ratios the indomethacin sediments entirely with liposomal ~tructure~ at the top of the 570 sucro~e 8radient.
Therefore, in all Rubsequent studies higher pho~pholipid/indomethacin ratios of about 6:1 by weight or about 2.6:1 molar ratios were employed to insure complete encapsulation of drug with the corresponding primary absence of exogenou~ nonsteroidal anti~inflammatory drug. ~atios of about 50:1 b~ weight are al~o prefered with ratios of 20:1 being most preferred.

It i9 to be understood that Pach nonsteroidal anti-inflammatory therapeutic agent will have a particular optimal lipid:drug molar and weight ratios in preparations primarily ab~ent exogenou3 therapeutic agent. Simple t~ting via 3ucro~e gradient or other appropriate tests will delimit the lipid:drug ratio~ appropri~te for any reaction conditions to produce liposome preparations primarily absent of exogenous nonsteroidal anti-inflammatory therapeutic agents. So long as an effective liposomally encap~ulated therapeutic dosage may be administered while exogenous nonsteroidal anti-inflammatory therapeutic agent is primarily absent by not bein8 present at levels greater than about 30% by weight ulceration and gastric irritatlon will be substantially reduced.
This limitation a~ ~o exogenous nonsteroidal antl-inflammatory i9 a critical limitation of this in~ention. A therapeutically effective dosage of nonsteroidal anti-inflammatory therapeutic agent i9 underRtood to be that dosage at which the desired physiological response is exhibited.

A9 to indomethacin in the preferred embodiment no more than about 25% exogenous dosages administered at about 0.5 to 4 mg/kg are utilized.
In generall the more limited the exogenous non3teroidal anti-inflammatory therapeutic agent the less prevalent gastrointe~tinal irritation and gross eastrointestinal insult. Each nonsteroidal an~i-inflammatory therapeutic agent will present a different efficiency of capture within particular lipo~omes. Where liposomal capture i~ insufficient to avoid excessive e~ogenous non~teroidal anti-inflammatory therapeutic agent, liposome preparation~ may be washed.

The liposome preparations of this invention may be washed of excess e~ogenou nonsteroidal anti-inflammatory therapeutic agent by centrifugation of the preparation for sufficient time and at sufficient velocity to cau~e lipo~omes to sediment but not to lose integrity. The supernatant i3 decanted and the liposomes are re~u~pended in saline or other aqueous media. This procedure may be repeated until sufficient exogenous non~teroidal anti-inflammatory therapeutic agent is eliminated.

,.. :.,, ,., , : ~

Other means of removing excess exogenous nonsteroidal anti-inflammatory ~herapeutic agen~ such as dialy~is or gel filtratlons are ~uitahle and well known in the ar~.

The compo~ition3 and methods of this invention are useful in reducing the adverse reactions to mam~als including humans arising from tre~tmen~ with nonsteroidal an~i-inflammatory therapeutic agent. Some measure of thi3 effect is made by acute and chronic ulc2ration teqts as described below.

Gastric ulcers in rats (Wistar) can be acutely induced by oral or subcutaneous admini~tration of nonsteroidal anti-inflammatory therapeutic agents such as indomathacin to animals pre~iou~ly starved for about 18-24 hours. For oral studies, it i9 convenient to use indomethacin but other nonsteroidal anti-inflammatory therapeutic agent can be similarly employed. Thi~ model i9 also useful in examining other nons~eroidal anti-inflam~atory therapeutic aBent~ and other mammal~. Simply varyin~
doses will induce gastric ulcer~ for other agents and mammals in this model. Unlike gastric ulcer protocols, it has been shown that for nonsteroidal anti-infl~nmatory effects such as indomethacin-induced intestinal lesion~ to occur, animals must be allowed free access to food ~ and water during the study.

The acute gastric ulcerative effects observed from adminiqtrstion P.O. to rat~ of variouq dosage~ of indomethacin in P~G-400 compared to indomethacin encapsulated within liposomes that were not gastric resi~tant ~CHSt and THSt) ("t" denotes the tris salt~ and in gastric resi~tant lipo~omes (egg phosphatidylcholine MPV9) are lllu~trated in Table 3. Substantial dose responsive ulceration occured when drug was completely solubilized into PEG-400 ~ehicle. An average 25 mm total ulcer length was obtained at the top dosage of 10 mg indomathacin/kg body weight. In individual e~periments this number has reached a high a~ 45 mm. In liposome preparations indom~thacin was fully dissolved into the membrane, and, unliXe PEG-400 ~ehicle, aqueous ~olutions of gaRtric re~i tant indomethacin-EPC MPV3 which primarily did not carry exogenous -23~

,.. .
~ .

' , , :

drug were capable of imparting complete protection at the lowe3t dosage of drug and over 75~ protection at higher dosage~ in chronic admini~tration. Indomethacin-C~St liposomes or indomethacin-THSt liposomes, both sen3itive to acid condltion~ in the GI tract, did not provide a~ significant ga~tric prot~ction at the highest dose of drug.
Lipids such as CHSt lipoqome~ or indome~hacin-THSt liposomes, both ~ensiti~e to acid conditions in the GI tract, did not provide as significant ga~tric protection at the highe3t dose of drug. Lipids ~uch a~ CHSt (cholesterol hemisuccinate tris) and THSt (tocop&erol hemi~ucclnate tris) that typi~y the cla~s of lipids that are not gastric resistant.

As seen from Table 4, ~ubstantial inte3tinal ulceration i~ evident following repeated admini3tration of indome~hacin in PEG-400.
Sign~ficant inhibition of ulcer formation i9 evident at 4 mg/kg oral do~ag~ of drug when EPC MP~ encap~ulated 19 admini~tered over the same 4-day schedule. As was observed in acute ga3tric ulcer experi~ents, a lack of protective action iq also noted in the inte~inal model sy~tem when indomethacin is incorporated into elther CHSt or THSt lipo~omes.

The protection afforded by gastric resistant liposomal (MPV) encapsulation of drug i~ clearly highlighted in compari~on to drug in PEG-400 when intestinal lesions are quantltated after chranic dosing ~Table 4). Table 4 further illustrates that protection from ulceration afforded by saturated lipid gastric resi~tant liposomes i9 at least equivalen~ to unhydrogenated liposomes.

The "controlled relea~e" of the encapsulated nonsteroidal anti-inflammatory therapeutic agent of ~uch liposome~ sub~tantially compri~ed of ~aturated lipid i9 disclosed in FIG. 2. The "controlled release" pattern of hydrogenated soy phosphatidylcholine i~ of ~uch level that, via oral dosage9 some liposome3 will be excretgd prior to delivery of all therapeutic agent to the receivin4 animal. Thl~ factor must, of cour~e, be taken into accoun~ when ~electing the dosage to be adminiYtered such that ~ufficien~ therapeutic agent will be elaborated, ~ ' .
.

0 ~ 3 ~
prior to excretion of the liposomes, to yield the desired availability of therapeutic agent in the ga3trointestinal tract for uptake by the animal.

Each nonsteroidal anti-inflam~atory therapeutic agent will have a do~e re3ponse curve below which chronic admlnistration of therapeutic agent produces no gross damage such as ulceration and abo~e which liposomal encapsulation and primary absence of exogenous therapeutic agent will not afford complete protection. However, this invention is directed to facilitate administration and particularly chronic admini~tration (about 3 or 4 day or longer) of nonsteroidal anti-infla~matory therapeutic a8ent by reducing gastrointestinal irritation, and by avoiding or reducing ulceration. For indomethacin daily oral dossgeQ below about 2 mg/kg generally produce no gro~s ulceration but may produce gastrointestinal irritation. Notwithstanding thi~ general case> oral human doses of indomethacin used in this invention are substantially similar to those used in unencapsulated preparations and are usually about 1 me/kg. These do3es may produce gastrointestinal ulceration in humans. The dosages referred to herein are in terms of therapeutic agent that will be available for uptake after release from the encapsulating liposomes. In the case of llpo~omes substantially comprised of hydro~enated lipid, the liposome may be excreted prior to full release of therapeutic agent. This factor must be ta~en into account in selecting the dosage to be ad~inistered.

Chronic administration of nonsteroidal anti-inflammato~y therapeutic agent is a well known method of use of such agent~. Chronic administration, for example in arthritis treatment, may be from as short a period as about 3 or 4 dAys or for a~ long as for the life of the recipient.

In pharmaceutlcal preparations, lipo~omes con~aln~ng nonsteroidal an~i-infla~matory therapeutic agent are suspended in an accep~able pharmaceutical diluent or carriers such as water or saline or other ~uitable carriers or diluentsO A typical preparation comprises from about 1 to 10 ml of aqueous ~olu~ion containing a therapeutically ' : .

1~2013~
effecti~e dose. A typical liposome containin~ indomethacin preparation i9 abo~t 25 mg indomethacin at least 70% liposomally encapsulated in about 5 or 6 ml aqueous media.

NSAIDs are generally lipophilic, and partition within the lipid portion of the liposome which in turn may be in a~sociation with a suitable pharmaceutical carrier. The proportional ratio of active ingredient to carrier will naturally depend on the chemical nature, solubility, and ~tability of the ac~ive ingredient, as well as the dosage contemplated. For the oral mode of adminlqtration, an NSAID-liposome compoQition of this invention can be used in the for~ of tablets, capsules, lozenges, troches, powders, ~yrup~, elixirs, aqueous solutions and suspenqions, and the llke. In the case of tablets, carriers which can be used include lactose, qodiu~ citrate, and salts of phosphoric acid. Various disintegrants such as starchJ and lubricating agents such as magnesium ~tearate, ~odium lauryl sulfate snd talc, are commonly used in tablets. For oral administration in cap~ule form, useful diluent~ are lacto~e and high molecular weight polyethylene glycols. When aqueous ~uspensions are required for oral use, certain sweetening and/or flavoring agents can be added. For parentexal administration or in~ection via intravenous, intraperitoneal, intramuscular, subcutaneous, intra-aural or intra- mammary route sterile ~olutions of the ~SAID-liposome composition are prepared and the pH of the solutions are suitably ad~uqted and buffered. For intravenous u3e, the total concentration of solutes should be controlled to render the preparation i~otonic.

In another example of their use, vesicle-entrapped compound~ may be incorporated into a broad range of topical dosage forms including but not limited to gelsJ oils, emul3ions and the like. For lnstance, the su~pension containing the entrapped compound may be added to ~he aqueous phase a} an ingredient in any type of liposome preparation (e,~., SPLVs, ~PVs, FATMLVs, MLVs, SUV9, L W q, REVs and others). This allows the entrapment of the wAter-insoluble compound in the pho pholipid liposomes. Such preparation~ may be admini3tered as topical creams, ' 1~20130 pastes, ointments, gels, lotions and the like for direct application to the inf]amed area.

NSAlDs are generally administered to humans in dosages ranging from about 20 mg to about 3200 mg a day depending on the NSAID. For example, indomethacin dosage ranges from 50~200 mg/day. Ibuprofen dosage ranges from 1200-3200 mg/day. The actual dosages should generally be determined by a physician. Similarly, other mammals such as horses may be administered these compounds in dosage ranges of 2 mg/kg/day - 800 mg/day (regardless of weight). For example, naproxen dosage for horses is 10 mg/kg/day in two divided dosages when given by oral route of administration. The prescribing physician or veterinarian will ultimately determine the appropriate dose for a given subject, and this can be expected to vary according to the age, weight and response of the individual subject, as well as the nature and severity of the subjects' symptoms.

The liposomes oE the present invention may be dehydrated, thereby enabling storage for extended periods of time until use. Standard freeze-drying equipment or equivalent apparatus may be used to dehydrate the liposomes. Liposomes may also be dehydrated simply by placing them under reduced pressure. Alternative]y, the ]iposomes and their surrounding medium can be frozen in ]iquid nitrogen prior to dehydration.
Dehydration with prior freezing may include the presence of one or more protective sugars in the preparation, according to the process of Janoff et al., PCT Patent Application No. 85/01502, pub]ished February 27, 1986, entitled "Dehydrated Liposomes". Examples of protective sugars that may be used include but are not limited to treha]ose, maltose, sucrose, glucose, lactose and dextran. Alternatively, multilamellar vesicles may be dehydrated with prior Ereezing without protective sugars. When the dehydrated liposomes are to be used, rehydration is accomplished by simply adding an aqueous solution, e.g., distilled water, to the liposomes and allowing them to rehydrate.

D

1~2~30 The liposomes of the present invention may also be remote loaded with ionizable agents according to the disclosure of Bally et al., Canadian Patent No. 1,270,198 issued June 12, 1990, entitled "Encapsula-tion of Antineop]astic Agents in Liposomes". In this procedure, a transmembrane potential is created across the bilayers of the liposomes during formation, and the ionizable agent is loaded into the liposomes by means of the transmembrane potential. This potential is generated by creating a concentration gradient for one or more charged species (e.g., Na~, K+ and/or H+~ across the liposome membranes. The concentration gradient is created by producing liposomes having different internal and external media, i.e., internal and external media having different con-centrations of one or more charged species. The ]iposomes may be dehy-drated prior to or following loading with agent.

The present invention decreases the u]cerogenic effect of NSAIDs, and may improve the efficacy of such drugs. In addition, such protection may be afforded by the liposomes of the present invention against ulcers produced by stress or alcohol consumption.

In the ulcer protection embodiment of the present invention, u]cerogenic activity of Eree indomethacin was compared to that of liposome-entrapped indomethacin as wel] as liposomes substantially free o exogenous indomethacin.

In the anti-inflammatory bioactivity embodiment of the present invention, efEicacy oE the liposome-drug preparations was measured in some embodiments by the edema intensity of a paw previously injected with an edema producing amount of carageenan.

Reduction of edema fol]owing administration of free NSAID such as indomethacin was compared to that following treatment with liposome-entrapped indomethacin.

As may be seen from the foregoing description, therapeutically effective doses of nonsteroidal anti-inflammatory therapeutic agents may .~ I .

,. :. .

~ 3~13~
be encapsulated in gaQtrlc resistant liposomes yet be substantially reduced in gastroin~estinal irritation usually associated with nonsteroidal an~ inflammatory therapeutic a8ent regimens. The reduction in ga~tric irritation i~ mos~ marked in chronic administration of nonsteroidal anti inflammatory therapeutic agentq in excess of about three or four days.

Therapeutic regimens will vary considerably ba~ed in part upon the mammal bein~ medicated, the condition being treated, and the particular nonsteroidal anti-inflammatory being utilized and whether or not the liposomes w~ll fully release the ~herapeutic agent prior to excretion.
While no exact limit~ may be placed upon nonsteroidal anti-inflammatory therapeu~ic regimens of treatment the regimens will often extend fro~
about days to year~ with dose3 about 3 time~ per day of nonsteroidal anti-inflammatory therapeutic agents from about 0.1 ~o 10 mg/kg (as released). The gastric resi~tant lipo~omes of this invention are most conveniently administered orally ~uspended in any ~uitable pharmaceutical carrier as an oral medication, however without limitation subcutaneous, intravenou~, and intraperitoneal adminiqtration and other known methods of administration are also contemplated. Howe~er, notwithstanding these general parameters, each therapeutic regimen may be individually determined by a physician in view of many factors including age, physical condition of patient and condition bei~g treated. Further, while ulceration or irritation may not be fully avoided, it will be reduced.

PREPAR~TORY STEP 1 LTPID PURI_ICATION

Digalactosyl diglyce~ide (DGDG) was obta~ned co~mercially or was prepared from fresh ~pinach leave~ according ~o $he following procedure:

One hundred grams of spinach leave~ were chopped into 1 cm pieces and placed in a Waring blender ~it~ 300 ml of iRopropanol at 70-80C.

`

the mixture was blended on high ~peed for two minutes. The resulting 31urry was filtered through two layers of Whatman #l filter paper and the re~idue washed wi~h 200 ml of the hot isopropanol. The re3ulting filter cake was placed in the blender with 200 ml chloroform:isopropanol (1:1 v/v) and blended as above. The resulting homogenate was filtered as above and the residue ~ashed with 200 ml of chloroform:isopropanol (1:1 v/v~ and then 200 ml chloroform. The filtrate was rotoevaporated in vacuo to a lipid film. The film was then dissolved in 200 ml chloroform, and the solution wa~hed three times with 100 ml of 1% (weight:volume) sodiu~ chloride aqueous ~olution in a separa~ory funnel. The or~anic phase was separated and 5 ml benzene were added to the organic pha~e.
The organic solvent was removed in vacuo to produce a film. The film wa~
resuspended in 10 ml benzene and the 3eparation and solvent removal steps were repeated. The film was stored ~uQpended in 25 ml of chloroform.

The digalactosyl diglyceride was purified from the above film suspen~ion by the following procedure: Fifteen grams of salicic acid that had been activated by bakin8 at 100C for three hours was combined with 50 ml of chloroform. The salicic acid 31urry was packed into a 20 cm X 40 cm column and the bed washed twice with chloroform. 175 g (5 ml) of the lipid Aolution wa~ loaded onto ~he column and the flow rate ad~usted to 3-5 ml per minute. 175 ml of chloroform was applied to the column, removing pigments in 12 ml fractions; followed by 70 ml of chloroform:acetone ~1:1 Y/V) applied to the column; followed by 700 ml of acetone. The flrst (5) 12 ml fractions contain MGDG, followed by DGDG in fractions 9-14J and finally phospholipids, in the remaining fractions which were discarded. Purity of the DGDG was assayed using thin layer chromatography ("TLC"), accordlng to the procedures of Rouser, et al.
(Lipid Chromatographic Analysis, Dekker Inc., New York, 1, pp. 99-162, 1967).

Fractions containing DGDG, as determined by TLC were combined and rotoevaporated under reduced pres3ure to a film. ChloroforD (10 ml) wa~
added and the solution ~ran~ferred to a pre-weighed flask, and rotoevaporated under reduced pressure to a fllm. The flask containing . ,. ' ~

~013~
lipid film wa~ agaln weighed and the difference calculated to be the weight of the lipid.

The presen~ invention is exemplified by the following Example~, bu~ the ~cope of the invention i~ not limited thereto.

PR~PARATION OF A NO~STEROIDAL_ANTI-INFLAMMATORY DRUG COMPOSITION

Digalactosyl diglyceride (DGDG~ (500 mg~ (obtained from Serdary Reqearch Laboratories, London, Ontarlo, Canada) in chloroform at 5 mg/ml was combined with 25 m~ of indomethacin in a round bottom flask. The chloroform was removed by evaporation under red~ced pres ure. Diethyl ether (5 ml~ was added to the re~ulting lipid-drug fllm and the film resuspended. The fla~k was placed ln a bath sonica~or and 1.0 ml of PBS
pH 7.4 wqs added. The ~olvent waq removed under a nitrogen ~tream while ~onicating. The resulting llpld-drug pAste was rehydrated with 2.5 ml PBS at pH 7.4. Resulting lipo~omes contained 10 mg/ml indomethacin.

INDUCING INTESTI~Q~_ULCERAT~_N (ACUTE) Twenty 225-250 g male Wistar rats were ~tarved for 18-24 hours prior to dosin~. ~ats were allowed acces~ to water throughou~ the study. Ten rats in the free drug control group were then administered one oral dose of indomethacin, dlssolved in polye~hylene glyc~l 400 at 7 mg~ml and administered at 10 mg/kg body weight of l~domethacin.

Four hours following dosing, rat~ were sacrlficed by carbon dioxide ano~ia and their ~tomach~ surgicAlly removed by sever~ng at the cardiac and pyloric sphinctors. The stomachs were opened alon~ the le~ser curYe, flattened, and washed with aline. Ulcer lengths were counted under a di~secting microscope (Amerlcan Optical9 Buffalo, ~Y) ~31~

: ~ -.

3~130 equipped with an eyepiece micrometer, and lengths were summed and averaged for all animals in a single treatment group.

Ulcer protection was as~essed by summing and averaging ulcer lengths (in mm) of the treated group and comparing that value to that of the group admlnistered free drug (Example 3~. Percent ulcer inhibition was calculated by dividing the average length of ulceration of the liposome-treated group by that of the corresponding free drug group, and multiplying by 100.

~XAMPLE 2 ADMINISTRATION OF DGDG LIPOSOMES

The procedures and materials of Example 1 were employed using 10 mg/kg body ~eight of indomethacin entrapped in stable plurilamellar vesicles (SPLVs) composed of digalactosyl diglyceride; ulcer protection was assessed as in Example 1. Table 1 demonstrates that the oral administration of indomethacin in DGDG liposomes ameliorate3 the ulcerative activity of indomethacin, as compared to that of free indomethacin.

ÇONTROL 2 INFL~M~TION ~D~ Q~

Eight female Wi~tar rats weighing approximately 100 grams were allowed free access to food and water. A tatoo line wa~ inscribed onto the right rear paw ~ust below the hair line. Initial paw volume was measured by a transducer-linked plethysmometer (Stoelting Co., Chicago, Illinoi~) which contained a saline solution. The lnstrument measures paw volume~ by an electrical charge difference across two electrodes resulting from the volume displacement by the paw. A tr~nsducer corrects thi~ charge difference into cu~ic centimeter~ of volume displaced.
Following immersion of ~he paw in the perspex cell to the inscribed line~
a direct measurement of the displacement volume was recorded.

~32~130 Rats were orally dosed with 2 mg/kg body ~eight of indomethacin in polyet~ylene glycol 400.

At 30 mlnutes po~t treatment, rats received an in~ection of 0.1 ml of 1.5% carageenan in saline (l.OM NaCl) directly into the rear paw pad.
The paw volume was again determined 2.5 hourq after the carageenan admini~tration. The edema intensity (EI) was calculated:

Edema Intensity = Final DaW volu~me -_initial paw volume Initial paw volume and averaged for the eight rat~.

The paw volumes for the eight rat~ were averaged and the percent swelling inhibition was calculated:

% Swelling Reduction:

Untreated control avR? EI - Treated grouD av~,_EI X 100 Untreated control avg. EI

~XAMPL~ ~

INFL~LTION REDUCTION

Th~ procedures and materials of Example 2 were employed using

2 mg/kg o~ indomethacin entrapped in SPLVs made with DGDG. Rat~ were oraIly dosed with the lipo~omes, and percent swelling reduction was calculated a~ in ~xample 2. Table 2 shows the comparabie swelling reduc~ion of free indomethacin (Example 2) and DGDG-lipo~ome entrapped indomethacin in th~ acute carageenan p8w edema model.

.
:
- . : :

, ` 132~13~
Example 4 EIPOSOME PREPARATION

Preparation of a nonsteroidal anti-inflammatory therapeutic agent comprising 200 mg of lipid was dissolved into 5 ml of ethanol. To that solution wa~ added 10 mB of indomethacin. To the resulting ~olution 0.6 ml of saline was added and the solvent then was removed by rotoevaporation, leavin8 a film.

The film wa~ resuspended thus for~ing liposome~ at the desired concentration of indomsthacin. For the lipo30me~ of Control 3 the material wa~ brought up to 2 ml final volume and for Control 4 the material was brought up to S ml.

Example 5 PREPA~ATIO~ OF SPLV LIPOSOMES ($TABLE VESICLES) In a flask 18.48 gm of indomethaci~ and 369.6 gm egg phosphntidylcholine were dissolved in 1848 ml methylene chloride at room temperature and the sample pressure chloride. To that was added 1,108 ml saline. The lipid phase was removed while stirrlng under vacuum until solvent i~ removed. To the re3ulting material wa~ added saline to a final volume of 3.186 liters. This preparation wa~ then ready for administration.

Exam~le 6 TEST ~ROCEDURE : LIPOSOI~ L_E~ P~D/
EXOGE~OUS ~O~STEROID~L A~TI-INF~AMMATORY THERAPEUTIC AGE~T

In thi3 example, 0.25 uCi of 14C-indomethacin were included in 100 =g preparation~ of lipo~ome containing variou~ly 15 ~o 25 mg of ~: '' '' ' ' - ' ' ~32~130 indomethacin prepared by the method of Example 4. The aqueous pha~e of the liposomes wa~ comprised of 0.3 ml aliquots of Ringer saline solution containing 10 mM of calcium. The~e aliquots were then loaded onto continuous 10 mll 5-20% sucrose Bradients and centrifuged at 288,000 xg for 2.5 hours. The specific re~ults of this test are shown in Figure 1.
The specific conditions for determining percentages of exogenou3 nonsteroidal antl-inflammatory therapeutic agent by this method or by o~her methods as well known to tho~e ~killed in the art.

Example 7 WASHING LIPOSOMAL PREPARATIONS

The liposome preparation of ~xample 6 washed 3 times with 5 ml saline after one hour of centrlfugation at 25,000 g retains less than 5%
exogenou~ indomethacin.

Control 3 INDUCING GAS2RXC ULCERS CACUTE~

Indomethacin at dosages between about 2 mg/kg and 10 mg/kg were given in 0.5 ml of neat PEG-400, vehicle or as incorporated into the liposomes at a 200 mg/kg dosage of phospholipid. Four hours following oral administration of test formulations, animal~ were sacrificed by CO~ ano~ia and the stomachs were excised. For systemically-induced gastric ulceration, dosages of PEG-400 solubilized indomethacin between 4 mg/kg and 50 mg/kg in 0.5 ml were administered subcutaneously. For subsequent e~periments a dosage of 30 mg/kg was found to induce the maximum respon3e. Tissue wa~ thoroughly rinsed in ~aline and the inner mucosal surface was laid fl~t for micro~copic evaluation. Visible ulcers were ~uanti~sted using a dissecting miocro3cope (AO Optical, Buffalo, ~Y) - . . . , ~

- ~ , ', ' ' .~ , .

~320~30 equipped with an eyepiece mlcrometer. Re3ults using this test were expressed in millimeters of ulceration and ~n percent inhibition of ulcers where treatment was effected. For all studies, a minimum of 10 animals per experimental group were employed. Experiments were conducted in triplicate, thereby representing a minimu~ of 30 rat~ per group.

Control 4 I~DUCING I~TESTINA~ ULCERATIO~ ~ÇHBONIC) Single daily oral dosages o indomethacin at about 4 mg/kg in vehicle or in monophasic ve~icle~ were administered over 4 days. Test animal~ in this example were rats (Wistar). On the fifth day the full length of the inte~tine~ were excised and ~lit open opposite to the attached mesenteric issue. The mucosal surface was rinsed wl~h saline, the ulcers (including perforations) were quantitated in m~lllmeters as described about or by determination of total surface area of lesions.
Thi~ was accomplished by means of a planimeter (Zida~, Carl Zeiss, West German~) optically linked to an SR stereo dissecting microscope (Zeiss) with a draw tube attachment. In this example re~ults were expressed as total ~m2 of ulcerated ~urface and as percent inhibition of ulceration when treatment was provided. Average values were calculated ba~ed upon a minimum of 5 animals p~r group from at least duplicate experiments. A
higher mortality rate (20-30%) was observed in rats receiving indomethacin solubilized in vehicles at this dvsage then in liposome-treated ~roups. Sham vehicle, however, were nontoxic to the animal~. Clearly this method is easily adaptable by thoqe skilled ln the art ~o other nonsteroidal anti-in~lammatory therapeutic agent~ and other animals.

:, ,, - -, ~

EXamD1e 8 13 2 013 0 PREPARATION OF AND DRUG RELEASE WITH HYDROGENATED LIPOSOMES

Three formulations for comparison of saturated and un~aturated lipo~omes were prepared by the method of Example 5, each containing S indomethacin as the therapeutic agent. These were lipo~omes made from (a) 5 gm of EPC and 100 mg of indomethacin, (b) 5 gm of hydrogenated 90Y
phosphatidylcholine and 100 mg of indomethacin, and (c) 3.35 ~m hydrogenated soy phosphatidylcholine, 1.645 gm cholesterol and, 100 mg indomethacin.

The resulting liposomes were administered orally to rats at a dosage of 10 mg/k~ and blood levels of indomethaci~ determined over a 24 hour period by high pressure llquid chromatography. The results shown in FIG 2 discloses a distinc~ blood level profile for release of nonsteroidal anti-inflammatory therapeutic agent into the blood after oral admin~tration in gastric reQi~tant liposomes. The hydrogenated soy pho~phatidylcholine displays a "controlled release" ab~ent the plasma peak characteristic of other lipo30mes of less saturation. Thi3 pattern of release indicates that some liposome~ will be excreted prior to delivery of all therapeutic agent which must be taken into account in ~electin~ the dosage to be admini~tered to yield the dosage taken up from the ~a~trointestinal tract.

The foregoing description is merely illustrative of the invention. Other examples of the invention will be immediately obvious to those skilled in the art. It is to be particularly understood that any non3teroidal anti-infla~matory, alone or in combination with others, may be used a~ primarily encapsulated in an~ ga~ric re3i3tant liposome.

.:, ' ' ', . :
- . . .

t3~013~

, I~DOMETHACIN mm % ULCER
TREATMENT DOSAG~ (m~/k~)ULCERATIONINHIBITION
Free Indomethacin in Vehicle 10 43.9 Indomethacin-DGDG
SPLVa 10 1.1 97 . 5 _ _ INDOMETHACIN % SWELLING
TR~ATMFNT DOSAG~ tmRJ~gl REDUCTION
Free Indomethacin 2 37.2 Indomethacin-DGDG-SPLV 2 36.9 ~32~3~

COMPARISON OF GASTRIC UL~ERATION PRODUCED FROM ACUTE
ORAL ADMINISTRATION OF IMDOMETHACIN
IN VEHICLES OR LIPOSOMES
_ _ . . . _ YO Ulcer Inhibition Indomethacin mm Rela~ive to TreatmentDosa~e_fm~/k~3 Ulceration ~ SEM* PEG-400 Control Indomethacin in 2 1.1 i 0-3 PEG~400 4 3.4 ~ 0.8 6 9.7 ~ 1.2 8 22.3 i 3.4 25.3 i 2.6 Indomethacin-EPC 2 0 100 Liposo~es 4 0~8 i 0-3 76.4 6 1.8 i 0.6 81.4 8 3.3 ~ 0.9 ~5.2 ~0 3.6 ~ 1.1 85.7 Indomethacin-CHSt 10 28.7 ~ 4.8 (-)13.4 Liposome~

Indomethacin-THSt 10 19.4 i 4.4 23.3 :
` *Standard error of the mean , : -39=

~. :

:

~32~130 CHRONIC ORAL DOSING OF INDOMETHACIN
IN BITHER V~HICLES OR LIPOSOMES
_ % Ulcer Inhibition Indomethacin ~m2 Rela~ive to Vehicle ~ _tm~/k~)Ulceration ~ SEM PEG-400 Con~rol PEG-400 4 759.3 ~ 120.6 EPC-Lipsomes 4 91.4 ~ 35.4 86.1 CHSt Liposomes 4 1084.20 i 160.6 (-)64.5 THSt Liposomes 4 398.6 i 230.7 39.5 ~SPC Lipo~omes 4 80.7 ~ 42.8 87.7 : :

: ~ :
~ 40-,

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A composition comprising a therapeutically effective amount of at least one nonsteroidal anti-inflammatory therapeutic agent primarily encapsulated in a gastric resistant liposome and primarily absent exogen-ous nonsteroidal anti-inflammatory therapeutic agent optionally wherein the composition additionally comprises a pharmaceutically acceptable car-rier or diluent.

2. The composition of claim 1 wherein the gastric resistant lipo-some is substantially comprised of a saturated lipid.

3. The composition of claim 2 wherein the lipid is hydrogenated egg or soy phosphatidylcholine.

4. The composition of claim 1 wherein the gastric resistant lipo-some comprises egg phosphatidylcholine.

5. The composition of claim 1 wherein the gastric resistant lipo-some comprises a glycolipid.

6. The composition of claim 5 wherein the glycolipid is a glyco-sphingolipid or a galactolipid.

7. The composition of claim 6 wherein the galactolipid is digal-actosyl diglyceride or monogalactosyl diglyceride.

8. The composition of claim 1, 2, 3, 4, 5, 6 or 7 comprising a phospholipid, a sterol derivative, or a tocopherol derivative.

9. The composition of claim 1, 2, 3, 4, 5, 6 or 7 wherein the gastric resistant liposome is of substantially equal solute distribution.

10. The composition of claim 9 wherein the liposome is an SPLV, MPV or FATMLV.

11. The composition of claim 8 wherein the gastric resistant lipo-some is of substantially equal solute distribution.

12. The composition of claim 11 wherein the liposome is an SPLV, MPV or FATMLV.

13. The composition of claim 8 wherein the nonsteroidal anti-inflammatory drug is selected from the group consisting of acemetacin, alclofenac, azapropazone, benoxaprofen, benrilate, carprofen, cholinemag-nesium trisalicylate, diclofenac, diflunisal, etodolac, fenbufen, fenclo-fenac, fenoprofen, fentiazac, feprazone, flufenamino acid, flurbiprofen, glucametacin, ibuprofen, indomethacin, indoprofen, isoxicam, ketoprofen, magnesium salicylic acid, meclofenamic acid, mefenamino acid, methylsali-cylate, naproxen, niflumic acid, osmosin, oxaprozin, oxaphenbutazone, phe-nylbutazone, piroxicam, pirprofen, salicylamide, salicylic acid, salicylic acid acetate, salicylsalicylic acid, sodium salicylate, sulindac, supro-fen, tenoxicam, tiaprofenic acid, tolmetin, and zomepirac.

14. The composition of claim 9 wherein the nonsteroidal anti-inflammatory drug is selected from the group consisting of acemetacin, alclofenac azapropazone, benoxaprofen, benrilate, carprofen, cholinemag-nesium trisalicylate, diclofenac, diflunisal, etodolac, fenbufen, fenclo-fenac, fenoprofen, fentiazac, feprazone, flufenamino acid, flurbiprofen, glucametacin, ibuprofen, indomethacin, indoprofen, isoxicam, ketoprofen, magnesium salicylic acid, meclofenamic acid, mefenamino acid, methylsali-cylate, naproxen, niflumic acid, osmosin, oxaprozin, oxaphenbutazone, phe-nylbutazone, piroxicam, pirprofen, salicylamide, salicylic acid, salicylic acid acetate, salicylsalicylic acid, sodium salicylate, sulindac, supro-fen, tenoxicam, tiaprofenic acid, tolmetin, and zomepirac.

15. The composition of claim 10 wherein the nonsteroidal anti-inflammatory drug is selected from the group consisting of acemetacin, alclofenac, azapropazone, benoxaprofen, benrilate, carprofen, cholinemag-nesium trisalicylate, diclofenac, diflunisal, etodolac, fenbufen, fenclo-fenac, fenoprofen, fentiazac, feprazone, flufenamino acid, flurbiprofen, glucametacin, ibuprofen, indomethacin, indoprofen, isoxicam, ketoprofen, magnesium salicylic acid, meclofenamic acid, mefenamino acid, methylsali-cylate, naproxen, niflumic acid, osmosin, oxaprozin, oxaphenbutazone, phe-nylbutazone, piroxicam, pirprofen, salicylamide, salicylic acid; salicylic acid acetate, salicylsalicylic acid, sodium salicylate, sulindac, supro-fen, tenoxicam, tiaprofenic acid, tolmetin, and zomepirac.

16. The composition of claim 11 wherein the nonsteroidal anti-inflammatory drug is selected from the group consisting of acemetacin, alclofenac, azapropazone, benoxaprofen, benrilate, carprofen, cholinemag-nesium trisalicylate, diclofenac, diflunisal, etodolac fenbufen, fenclo-fenac, fenoprofen, fentiazac, feprazone, flufenamino acid, flurbiprofen, glucametacin, ibuprofen, indomethacin, indoprofen, isoxicam, ketoprofen, magnesium salicylic acid, meclofenamic acid, mefenamino acid, methylsali-cylate, naproxen, niflumic acid, osmosin, oxaprozin, oxaphenbutazone, phe-nylbutazone, piroxicam, pirprofen, salicylamide, salicylic acid, salicylic acid acetate, salicylsalicylic acid, sodium salicylate, sulindac, supro-fen, tenoxicam, tiaprofenic acid, tolmetin, and zomepirac.

17. The composition of claim 12 wherein the nonsteroidal anti-inflammatory drug is selected from the group consisting of acemetacin, alclofenac, azapropazone, benoxaprofen, benrilate, carprofen, cholinemag-nesium trisalicylate, diclofenac, diflunisal, etodolac, fenbufen, fenclo-fenac, fenoprofen, fentiazac, feprazone, flufenamino acid, flurbiprofen, glucametacin, ibuprofen, indomethacin, indoprofen, isoxicam, ketoprofen, magnesium salicylic acid, meclofenamic acid, mefenamino acid, methylsali-cylate, naproxen, niflumic acid, osmosin, oxaprozin, oxaphenbutazone, phe-nylbutazone, piroxicam, pirprofen, salicylamide, salicylic acid, salicylic acid acetate, salicylsalicylic acid, sodium salicylate, sulindac, supro-fen, tenoxicam, tiaprofenic acid, tolmetin, and zomepirac.

10. The composition of claim 8 wherein the nonsteroidal anti-inflammatory therapeutic agent is indomethacin.

19. The composition of claim 9 wherein the nonsteroidal anti-inflammatory therapeutic agent is indomethacin.

20. The composition of claim 10 wherein the nonsteroidal anti-inflammatory therapeutic agent is indomethacin.

21. The composition of claim 11 wherein the nonsteroidal anti-inflammatory therapeutic agent is indomethacin.

22. The composition of claim 12 wherein the nonsteroidal anti-inflammatory therapeutic agent is indomethacin.

23. The composition of claim 1, 2, 3, 4, 5, 6 or 7 wherein said exogenous nonsteroidal anti-inflammatory therapeutic agent comprises less than about 30% by weight of nonsteroidal anti-inflammatory drug present.

24. The composition of claim 8 wherein said exogenous nonsteroidal anti-inflammatory therapeutic agent comprises less than about 30% by weight of nonsteroidal anti-inflammatory drug present.

25. The composition of claim 9 wherein said exogenous nonsteroidal anti-inflammatory therapeutic agent comprises less than about 30% by weight of nonsteroidal anti-inflammatory drug present.

26. The composition of claim 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 wherein said exogenous nonsteroidal anti-inflammatory therapeutic agent comprises less than about 30% by weight of nonsteroidal anti-inflammatory drug present.

27. The composition of claim 1, 2, 3, 4, 5, 6 or 7 wherein said exogenous nonsteroidal anti-inflammatory therapeutic agent comprises less than about 5% by weight of nonsteroidal anti-inflammatory drug present.

28. The composition of claim 8 wherein said exogenous nonsteroidal anti-inflammatory therapeutic agent comprises less than about 5% by weight of nonsteroidal anti-inflammatory drug present.

29. The composition of claim 9 wherein said exogenous nonsteroidal anti-inflammatory therapeutic agent comprises less than about 5% by weight of nonsteroidal anti-inflammatory drug present.

30. The composition of claim 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 wherein said exogenous nonsteroidal anti-inflammatory therapeutic agent comprises less than about 5% by weight of nonsteroidal anti-inflammatory drug present.

31. The composition of claim 1, 2, 3, 4, 5, 6, or 7 which is for use in minimizing the gastrointestinal irritation associated with the administration of a nonsteroidal anti-inflammatory drug to a mammal.

32. The composition of claim 8 which is for use in minimizing the gastroinetestinal irritation associated with the administration of a nonsteroidal anti-inflammatory drug to a mammal.

33. The composition of claim 9 which is for use in minimizing the gastrointestinal irritation associated with the administration of a nonsteroidal anti-inflammatory drug to a mammal.

34. The composition of claim 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 which is for use in minimizing the gastrointestinal irritation associated with the administration of a nonsteroidal anti-inflammatory drug to a mammal.

35. The composition of claim 23 which is for use in minimizing the gastrointestinal irritation associated with the administration of a nonsteroidal anti-inflammatory drug to a mammal.

36. The composition of claim 26 which is for use in minimizing the gastrointestinal irritation associated with the administration of a nonsteroidal anti-inflammatory drug to a mammal.

37. The composition of claim 27 which is for use in minimizing the gastrointestinal irritation associated with the administration of a nonsteroidal anti-inflammatory drug to a mammal.

38. The composition of claim 30 which is for use in minimizing the gastrointestinal irritation associated with the administration of a nonsteroidal anti-inflammatory drug to a mammal.

39. The composition of claim 24, 25, 28 or 29 which is for use in minimizing the gastrointestinal irritation associated with the administration of a nonsteroidal anti-inflammatory drug to a mammal.

40. The composition of claim 1, 2, 3, 4, 5, 6 or 7 which is for use in the treatment of inflammation, pain or fever in a mammal.

41. The composition of claim 8 which is for use in the treatment of inflammation, pain or fever in a mammal.

42. The composition of claim 9 which is for use in the treatment of inflammation, pain or fever in a mammal.

43. The composition of claim 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 which is for use in the treatment of inflammation, pain or fever in a mammal.

44. The composition of claim 23, which is for use in the treatment of inflammation, pain or fever in mammal.

45. The composition of claim 26, which is for use in the treatment of inflammation, pain or fever in mammal.

46. The composition of claim 27, which is for use in the treatment of inflammation, pain or fever in mammal.

47. The composition of claim 30, which is for use in the treatment of inflammation, pain or fever in mammal.

48. The composition of claim 24, 25, 28 or 29 which is for use in the treatment of inflammation, pain or fever in mammal.

49. A method of preparing gastric resistant liposomes according to claim 1, 2, 3, 4, 5, 6 or 7 comprising a) forming a solution of at least one gastric resistant lipid in at least one organic solvent plus a first aqueous component containing therein at least one nonsteroidal anti-inflammatory drug in amounts sufficient for form a monophase;
b) evaporating organic solvent of the monophase at a temperature and pressure which maintains the monophase and facilitates evaporation until a film forms; and c) adding a second aqueous component to the film and agitating the second aqueous component with the film in order to resus-pend the film and to form lipid vesicles encapsulating said nonsteroidal anti-inflammatory drug.

50. The method of preparing gastric -resistant liposomes according to claim 8 comprising a) forming a solution of at least one gastric resistant lipid in at least one organic solvent plus a first aqueous component containing therein at least one nonsteroidal anti-inflammatory drug in amounts sufficient to form a monophase;
b) evaporating organic solvent of the monophase at a temperature and pressure which maintains the monophase and facilitates evaporation until a film forms; and c) adding a second aqueous component to the film and agitating the second aqueous component with the film in order to resus-pend the film and to form lipid vesicles encapsulating said nonsteroidal anti-inflammatory drug.

51. The method of preparing gastric resistant liposomes according to claim 9 comprising a) forming a solution of at least one gastric resistant lipid in at least one organic solvent plus a first aqueous component containing therein at least one nonsteroidal anti-inflammatory drug in amounts sufficient to form a monophase;
b) evaporating organic solvent of the monophase at a temperature and pressure which maintains the monophase and facilitates evaporation until a film forms; and c) adding a second aqueous component to the film and agitating the second aqueous component with the film in order to resus-pend the film and to form lipid vesicles encapsulating said nonsteroidal anti-inflammatory drug.

52. The method of claim 49 wherein the product of step c) is treated by removing excess exogenous nonsteroidal anti-inflammatory drug.

53. The method of claim 50 wherein the product of step c) is treated by removing excess exogenous nonsteroidal anti-inflammatory drug.

54. The method of claim 51 wherein the product of step c) is treated by removing excess exogenous nonsteroidal anti-inflammatory drug.

55. The method of claim 52, 53 or 54 wherein the removal of excess exogenous nonsteroidal anti-inflammatory drug is carried out by washing in an aqueous solution.